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Southern Hybridization - The name of this technique is derived from the following:
(1) the name of its inventor, E.M. Southern, and
(2) the DNA-DNA hybridization that forms its basis. It is also called Southern blotting since the procedure for transfer of DNA from the gel to the nitrocellulose filter resembles blotting. This technique has since been extended to the analysis of RNA (northern blotting) and proteins (western blotting); these names are only jargon terms, i.e., reverse of Southern being northern and so on, and do not reflect any functional or historical significance.
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In Southern hybridization, a sample of DNA containing fragments of different sizes is subjected to electrophoresis using either polyacrylamide or agarose gel. The DNA sample may either be subjected to mechanical shearing or to restriction endonuclease digestion in order to generate the fragments.
Agarose gel is useful in separating DNA fragments of few hundred to 20 kb in size, while polyacrylamide is preferred for smaller fragments. Very large DNA fragments of upto 1000-2000 kb are separated in agarose gel with pulsed electrical fields, or field inversion.
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The gel provides a complex network of polymeric molecules through which DNA fragments migrate, depending on their sizes, under an electric field since DNA molecules are negatively charged. Smaller molecules of DNA migrate relatively faster than the larger ones. Marker DNA fragments of known sizes are run in a separate lane; this permits an accurate determination of the size of an unknown DNA molecule by interpolation.
The gels are stained with the intercalcating dye ethidium bromide which, gives visible fluorescence on illumination of the gel with UV light; as little as 0.05 µg of DNA in one band can be detected by using this dye. This approach is useful when few DNA fragments with considerable length differences are to be separated and studied. This approach also separates the closed circular (supercoiled), nicked (relaxed) and linear configurations of a single DNA molecule.
In many situations, it is critical to detect and identify DNA fragments in a sample that are complementary to a given DNA sequence, e.g., to demonstrate the presence of the gene in question in transgenics, to detect and stuty RFLP (restriction fragment length polymorphism), etc. This is achieved by Southern hybridization in which the following steps are performed.
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1. The restriction fragments of DNA present in agarose gel (after electrophoresis) are denatured into single-stranded form by alkali treatment.
2. They are then transferred onto a nitrocellulose filter membrane; this is done by placing the gel on top of a buffer saturated filter paper, then laying the nitrocellulose filter membrane on the top of gel, and finally placing some dry filter papers on top of this membrane.
The buffer moves, due to capillary action, from the bottom filter paper through the gel carrying with it the denatured DNA present in the gel; the DNA becomes trapped in the nitrocellulose membrane as the buffer phases through it. This process is known as blotting and takes several hours to complete. The relative positions of the bands on the membrane remain the same as those in the gel and there is a minimal loss in their resolution (sharpness).
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3. The nitrocellulose membrane is now removed from the blotting stack, and the DNA is permanently immobilized on the membrane by baking it at 80°C in vacuo.
4. Single-stranded DNA has a high affinity for nitrocellulose filter membrane. (Note that RNA lacks this affinity). Therefore, the baked membrane is treated, with a solution containing 0.2% each of Ficoll (an artificial polymer of sucrose), polyvinylpyrrolidon and bovine serum albumin; this mixture is often supplemented with an irrelevant nucleic acid, e.g., tRNA (pretreatment).
This treatment prevents nonspecific binding of the radioactive probe (to be used in the next step) probably by attaching macromolecules to all the free binding sites on the membrane. Often the above mixture is included in the hybridization reaction itself.
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5. The pretreated membrane is placed in a solution of radioactive, single-stranded DNA or an oligodeoxynucleotide (a DNA segment having few to several nucleotides) called probe. The name probe signifies the fact that this DNA molecule is used to detect and identify the DNA fragment in the gel/membrane that is complementary to the probe.
The conditions during this step are chosen so that the probe hybridizes with the complementary DNA on the membrane to the greatest extent with a low nonspecific binding on the membrane; this step is known as hybridization reaction.
Usually, the initial hybridization reaction is carried out under conditions of relatively low stringency of hybridization to permit a high rate of hybridization; this is followed by a series of post hybridization washes of increasing stringency, i.e., higher temperature or, more commonly, lower ionic strength, with a view to eliminate the pairing of radioactive probe to related sequences and to allow only perfectly complementary pairing.
6. After the hybridization reaction, the membrane is washed to remove the unbound probes.
7. The membrane is now placed in close contact with an X-ray film and incubated for a desired period to allow images due to the radioactive probes to be formed on the film. The film is then developed to reveal distinct band(s) indicating positions in the gel of the DNA fragments that are complementary to the radioactive probe used in the study.
It should be kept in mind that electrophoresis of sheared or restricted DNA produces a smear in which the fragments are distributed in a continuum according to their size, and there are no distinct bands. The distinct bands are produced by the hybridization reaction of the selected probe with one or few fragment sequences present in the gel.
The Southern blotting technique is extremely sensitive. It can be used to map the restriction sites around a single copy gene sequence in any genome (even of man). It is used for DNA fingerprinting, preparation of RFLP maps, detection and identification of the transferred genes in transgenic individuals, etc.
Recently some new membrane materials, e.g., nylon membranes, have been developed which have the following advantageous features:
(1) They are physically robust in comparison to nitrocellulose filter membranes,
(2) both DNA and RNA become cross-linked to them by a brief exposure to UV light, which
(3) saves the time needed for baking in vacuo in the case of nitrocellulose membranes, and
(4) the same membrane blot, e.g., a membrane onto which DNA/RNA has been transferred from a gel and cross-linked by UV exposure, can be used for search with more than one probe after removing the earlier probe by high temperature washing or some other denaturing procedure; in other words, the nylon membranes are reusable.
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